Role of native soil biology in Brassicaceous seed meal-induced weed suppression

lable at ScienceDirect

Soil Biology & Biochemistry 40 (2008) 1689–1697

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Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lb io

Role of native soil biology in Brassicaceous seed meal-induced weedsuppression

L. Hoagland b, L. Carpenter-Boggs b, J.P. Reganold b, M. Mazzola a,*

a USDA-ARS, 1104N. Western Avenue, Wenatchee, WA 98801, USAb Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164-6420 USA

a r t i c l e i n f o

Article history:Received 31 August 2007Received in revised form 6 February 2008Accepted 7 February 2008Available online 30 April 2008

Keywords:BrassicaceousAllelopathyPythiumGlucosinolatesWeed suppressionBiological controlBrassicaceae

* Corresponding author.E-mail address: [email protected] (M. M

0038-0717/$ – see front matter Published by Elsevierdoi:10.1016/j.soilbio.2008.02.003

a b s t r a c t

Biologically based weed control strategies are needed in organic and low-input systems. One promisingpractice is the application of Brassicaceous seed meal (BSM) residue, a byproduct of biodiesel production.When applied as a soil amendment, BSM residue has exhibited potential bioherbicide activity. In thisstudy, tree fruit orchard soils were treated with various BSMs and the impact of Pythium on weedsuppression was examined in field and greenhouse studies. Although weed control obtained in responseto Brassicaceous residue amendments has been repeatedly attributed solely to release of allelopathicphytochemicals, multiple lines of evidence acquired in these studies indicate the involvement of a mi-crobiological component. Reduced weed emergence and increased weed seedling mortality were notrelated to BSM glucosinolate content but were correlated with significant increases in resident pop-ulations of Pythium spp. in three different orchard soils. Seed meal of Brassica juncea did not amplifyresident Pythium populations and did not suppress weed emergence. Application of Glycine max SM didstimulate Pythium spp. populations and likewise suppressed weed emergence. Application of a mefe-noxam drench to Pythium-enriched soil significantly reduced weed suppression. These studies indicatethat a microbial mechanism is involved in SM-induced weed suppression and that selective enhance-ment of resident pathogenic Pythium spp. can be utilized for the purpose of weed control.

Published by Elsevier Ltd.

1. Introduction

Growth in organic and sustainable agricultural production sys-tems has generated demand for compatible weed control strate-gies. Brassicaceous seed meal (BSM) residue, a waste product of theoil extraction process, can provide a local resource for supplementalnutrients (Hoagland et al., 2007), disease control (Lazzeri andManici, 2001; Mazzola et al., 2001; Zasada and Ferris, 2004; Maz-zola and Mullinix, 2005), and/or weed suppression (Brown andMorra, 1997). However, the mechanisms contributing to the ob-served BSM weed control remain unclear (Boydston and Hang,1995; Brown and Morra, 1997).

Decreased weed emergence has been repeatedly documentedfollowing soil incorporation of Brassicaceous crop and BSM residues(Boydston and Hang, 1995; Al Khatib et al., 1997; Brown and Morra,1997). The mechanism of weed suppression has been attributed toallelopathy, which is defined as the inhibitory effect of one plant ormicroorganism on another through chemical release from the donorto the environment (Kobayashi, 2004). Glucosinolate hydrolysis

azzola).

Ltd.

products are thought to be responsible for the weed suppression in-duced by Brassicaceous residues (Brown and Morra, 1997). The hy-drolytic enzyme, myrosinase, and water are required for glucosinolatehydrolysis. The type, concentration, and functionality of glucoinolatehydrolysis products vary among Brassicaceous species. Glucosino-lates are present in all Brassicaceous plant parts, but are most con-centrated in seed (Borek and Morra, 2005). If cold pressed, residualBSM retains glucosinolate content and viable myrosinase after seedoil extraction (Borek and Morra, 2005). Therefore, it is reasonable tohypothesize that glucosinolate hydrolysis products have a role in theweed suppression resulting from application of BSM.

Although weed suppression by Brassicaceous residues has longbeen attributed to glucosinolate induced allelopathy, there has notbeen a consistent relationship between observed weed suppressionand measured glucosinolate content. For example, significant plantsuppression has been observed with low glucosinolate contentBrassica napus residues (Boydston and Hang,1995; Brown and Morra,1996; Al Khatib et al., 1997). These authors suggested either effectiveaction by a relatively small amount of a specific but unidentifiedglucosinolate hydrolysis product, or that microbial degradationresulted in production of other inhibitory compounds. Some gluco-sinolate hydrolysis products such as ionic thiocyanate have biocidaleffects, and are used as the active ingredient in several commercial

L. Hoagland et al. / Soil Biology & Biochemistry 40 (2008) 1689–16971690

herbicides (Borek and Morra, 2005). However, these products controlweeds at effective ionic thiocyanate (SCN�) concentrations of 137–1366 kg SCN� ha�1, much higher than that found in BSM amendmentrates that have been found to be phytotoxic (Borek and Morra, 2005).Phytotoxicity has been observed at BSM amendment rates of 1000–4000 kg SM ha�1, with only 8.8–35.3 kg SCN� ha�1, assuming com-plete conversion to toxic hydrolysis products (Borek and Morra,2005). In addition, soil physical, chemical, and biological character-istics influence expression and longevity of allelochemicals underfield conditions (Inderjit et al., 2001).

Incorporation of plant residue, including Brassica spp., is alsocommonly associated with rapid increases in total microbial ac-tivity, which can include plant pathogenic soil fungi and oomycetes(Grunwald et al., 2000; Manici et al., 2004; Cohen et al., 2005), withmany capable of inciting root, stem, or seed rots (Pitty et al., 1987)that can be fatal to both crop and weed species. Many members ofthe genus Pythium incite both pre- and post-emergent damping-offof plants. Populations of Pythium spp. in soil are amplified in re-sponse to organic matter addition, survive in competition withother microorganisms (Chen et al., 1988) and withstand frequentcultivation (Grunwald et al., 2000; Mazzola and Gu, 2000).

Application of Brassicaceous amendments may provide an al-ternative weed control strategy, but the mechanism of action mustbe better understood to generate guidelines and recommendationsfor use of this practice as a management tool. These studies wereperformed in or with multiple orchard soils to test the hypothesisthat induced amplification of resident Pythium spp. contributes tothe weed suppression observed in response to BSM amendments.

2. Materials and methods

2.1. Soils and soil treatments

Studies were conducted at or in soils collected from threeexperimental orchards: the Columbia View Experimental (CV) or-chard, Orondo, WA; the Wenatchee Valley College-Auvil Teachingand Demonstration (WVC) orchard, East Wenatchee, WA; and theTukey Horticulture Research and Experimental (TU) orchard, Pull-man, WA. Soils at these sites are characterized as Adkins very finesandy loam (coarse–loamy, mixed, mesic Xeric Haplocalcid) with1.3% organic matter (OM) and pH 7.6, Pogue sandy loam (coarse–loamy over sandy or sandy–skeletal, mixed, mesic AridicHaploxeroll) with 1–2% OM and pH 6.1–7.3, and Thatuna silt loam(fine-silty, mixed, mesic Oxyaquic Argixeroll) with 4–5% OM andpH 6.8, respectively. Plots at WVC and TU orchards are under or-ganic management.

Amendments used in field and greenhouse studies includeda low glucosinolate (glucosinolate content (GLC)¼ 21.8 mmol g�1)commercial rapeseed, B. napus cv. Dwarf Essex (Montana SpecialtyMills, Great Fall, MT), and two high glucosinolate mustard varieties,Brassica juncea cv. Pacific Gold (GLC¼ 303 mmol g�1) (Brown et al.,2004), and Sinapis alba cv. Ida Gold (GLC¼ 244 mmol g�1) (Brownet al., 1997). Nitrogen contents of the BSMs were 5.57, 6.09, and6.84%, respectively (Mazzola et al., 2007). Greenhouse experimentsalso included a non-glucosinolate containing soybean (G. max) seedmeal (no glucosinolate, 3% N) treatment and a pasteurized soiltreatment. All amendments were applied to soil at a rate of 0.3%vol/vol. All field and greenhouse experiments included a non-treated control. In 2005, the field experiment carried out at CVorchard included a 1,3-dichloropropene-chloropicrin (TeloneC17;DowElanco, Indianapolis, IN) soil fumigation treatment at282 l ha�1. A mefenoxam (Ridomil Gold EC 49% ai; Syngenta,Greensboro, NC) soil drench was used in the 2006 field experimentand all greenhouse experiments to selectively reduce plant in-fection by Pythium spp.

2.2. Greenhouse experiments

Composite soil samples were collected at WVC in spring 2005(WVC1), autumn 2005 (WVC2), and at TU orchards in spring2006 for use in greenhouse assays. Soil was also collected inspring 2006 from an experimental plot at CV orchard and anarea immediately adjacent with native (uncultivated) shrubsteppe vegetation. Ten soil samples were collected from withinthe root zone of random trees in established orchard sites toa depth of 10–30 cm, approximately 1–2 m from the tree baseand pooled. Soil was stored at ambient greenhouse conditionsuntil experiments were initiated. Three replicate soil samplesfrom each site/date were pooled and stored at 4 �C for sub-sequent laboratory analysis. For each experiment, soil was pre-mixed using a cement mixer and 2.5 l aliquots of soil wereplaced in 5-l tubs. Seed meal amendments were applied to soil intwo tubs per treatment, hand mixed and covered with lidsduring a 4 d incubation in the greenhouse at 22� 4 �C. At com-pletion of the incubation period, a composite soil sample wascollected from each treatment for laboratory analysis. At thesame time, mefenoxam was diluted to 0.635 ml l�1 and 116.7 mlwas applied to soil in one of the tubs representing each treat-ment. Soil from each tub was then placed in conical tubes(21�4 cm). Prior to planting, germination rates for each plantspp. were determined by placing 20 seeds onto moistened filterpaper in a petri dish, and counted after 48 h (Mazzola and Cook,1991). Subsequently, five Triticum aestivum (Wheat cv. Madsen)seeds, 10 Vicia villosa (Hairy Vetch) seeds, 10 Amaranthus retro-flexus (Pigweed) seeds, or seven Echinochloa crusgalli (Barnyard-grass) seeds were planted into conical tubes. Each seedtype� soil treatment combination was replicated in 10 growthtubes. Plants were individually watered when a dry soil surfacewas observed. Plant emergence was recorded at 5 d and again atharvest 21 d after planting. Twelve days after planting, threecones per seed type/soil treatment were randomly selected fordetermination of Pythium soil populations and root infection.

2.3. Experimental field plots

Field plots, 3.05 m2, were established at CV orchard in spring2005 and 2006 in a randomized complete block design with split-plots and five replicates. Seed meal amendments were applied at8533 kg ha�1, and incorporated to 15 cm depth using a rotovator.Forty-eight hours after BSM amendment, mefenoxam(0.635 ml l�1) aqueous solution was applied to half of each plot at1.48 ml m�2. In 2005, BSM was applied on 21 April and half of eachsplit-plot was split again and covered with a 152-mm thick clearplastic sheet (Sunbelt Plastics, Monroe, LA), which was removed on23 May (32 d). Plastic was not applied to plots in 2006.

In 2005, approximately 90 d after seed meal application, allshoot and root biomass was collected from each plot at CV orchardand divided into grass and broadleaf species. At the same site,aboveground weed biomass was also collected from a newlyestablished orchard planting employing the same soil treatmentsusing the same method. In 2006, 3 d following SM amendment, fiveT. aestivum seeds (cv. Madsen) were planted into each split-plot andgermination was recorded after 14 d. Forty days after amendmentapplication, four sub-samples (0.1 m2 each) of aboveground weedbiomass were cut and pooled for analyses within each split-plot. Allplant samples were oven-dried at 50 �C for 48 h and weighed todetermine dry biomass.

Soil samples were collected at 0, 3, 8 and 15 d post-BSMamendment. Three or four sub-samples were collected using a 2-cm diameter soil probe and pooled for analyses. Sampling depth of0–10 or 10–30 cm is indicated on all data in results. All soil sampleswere stored at 4 �C until analysis.

Table 1aEffects of seed meal amendments on percent emergence of Triticum aestivum whenestablished in two orchard soils

Treatment WVC1a WVC2 TU

5 d 5 d 21 d 5 d 21 d

Control 62 ab 62 c 62 bc 20 e 32 dControlþmefenoxam 82 ab 82 ab 90 ab 94 a 98 aPasteurized 94 a 94 a 96 a 96 a 92 aPasteurizedþmefenoxam 88 a 88 a 86 ab 64 cd 72 deB. napus 8 e 8 d 70 abc 4 f 18 deB. napusþmefenoxam 92 a 92 a 80 abc 92 ab 96 aB. juncea 68 bc 68 bc 64 bc 54 d 62 cB. junceaþmefenoxam 90 a 90 a 78 abc 78 cd 84 abS. alba 30 d 12 d 20 d 4 f 6 eS. albaþmefenoxam 90 a 90 a 70 abc 94 a 94 aG. max 12 e 12 d 54 c 2 f 8 eG. maxþmefenoxam 94 a 94 a 74 abc 88 ab 86 ab

a Orchard designations: WVC, Wenatchee Valley College Orchard, East We-natchee, WA, and Tukey, Tukey Horticulture Research and Experimental Orchard,Pullman, WA. WVC1 represents soil collected in spring 2005, and WVC2 representssoil collected in autumn 2005.

b Means in the same column followed by the same letter are not significantlydifferent (P> 0.05; n¼ 10).

Table 1bEffects of seed meal amendments on percent emergence of Vicia villosa when es-tablished in two orchard soils

Treatment WVC1a WVC2 TU

5 d 5 d 21 d 5 d 21 d

Control 13 bcdb 13 bcd 30 c 12 c 22 dControlþmefenoxam 23 ab 23 ab 49 a 23 abc 38 bcdPasteurized 10 cd 10 cd 47 ab 32 ab 64 aPasteurizedþmefenoxam 20 abc 20 abc 45 ab 17 bc 49 abB. napus 11 cd 11 cd 41 b 18 abc 37 bcdB. napusþmefenoxam 20 abc 20 abc 48 ab 22 abc 38 bcdB. juncea 20 abc 20 abc 18 d 11 c 26 dB. junceaþmefenoxam 25 a 25 a 45 ab 20 abc 40 bcS. alba 11 cd 11 cd 4 e 13 c 29 cdS. albaþmefenoxam 23 ab 23 ab 46 ab 28 abc 43 bcG. max 5 d 5 d 13 d 17 bc 29 cdG. maxþmefenoxam 17 abc 17 abc 47 ab 33 a 41 bc

a Orchard designations: WVC, Wenatchee Valley College Orchard, East We-natchee, WA, and Tukey, Tukey Horticulture Research and Experimental Orchard,Pullman, WA. WVC1 represents soil collected in spring 2005, and WVC2 representssoil collected in autumn 2005.

b Means in the same column followed by the same letter are not significantlydifferent (P> 0.05; n¼ 10).

L. Hoagland et al. / Soil Biology & Biochemistry 40 (2008) 1689–1697 1691

2.4. Characterization of soil and plant colonizing Pythiumpopulations

Three separate 5-g soil sub-samples from each field or green-house treatment were suspended in 25 ml sterile distilled water,vortexed 60 s and serial dilutions were plated on a Pythium semi-selective growth medium (PSSM; Mazzola et al., 2001). After 48 h,adhering soil was washed from plates under running water, andcolonies exhibiting typical Pythium morphology were enumerated.Hyphal plugs from representative Pythium colonies from each platewere transferred to new plates.

In greenhouse assays, composition of the Pythium populationrecovered from plant tissues was determined. Plants from eachgrowth tube were individually removed, rinsed with tap water, andsix root segments 3 cm in length were plated onto PSSM. In the potswhere no plants emerged, large weed seeds (T. aestivum and V.villosa) were extracted from the pot and plated onto PSSM. Pythiuminfection of each root/seed was recorded after 48 h.

Initial species identifications of Pythium isolates recovered weredetermined by DNA sequence analysis. Three 0.4 cm diameter plugswere excised from the growing margin of individual cultures,transferred to 5 ml 20%-strength potato dextrose broth, and in-cubated on rotating platform (150 rpm) at ambient laboratoryconditions. DNA was extracted from Pythium mycelium usinga MoBIO Ultraclean Soil DNA kit (Carlsbad, CA), and stored at�20 �C until analysis. Polymerase chain reaction amplification ofPythium DNA was conducted using the primer set internal tran-scribed spacer (ITS) 4 and ITS5 (White et al., 1990) in a GeneAmp9700 thermal cycler (Applied Biosystems, Foster City, CA) usingconditions previously described (Tewoldemedhin et al., 2006).Amplification products were confirmed by visual comparison toa 100 bp ladder following electrophoresis on a 1.5% agarose gelstained with ethidium bromide. Resulting amplicons were directlysequenced using a Dye Terminator Cycle Sequencing Quick Start Kitand a CEQ 8000 Genetic Analysis System capillary-based DNA se-quencer (Beckman Coulter, Fullerton, CA) with ITS1 (White et al.,1990) as the sequencing primer. Sequences obtained were com-pared with the online NCBI BLAST database.

Restriction fragment length polymorphism (RFLP) analysis wasalso employed to characterize the composition of Pythium spp.populations. ITS amplicons generated from each Pythium isolatewere digested individually in single enzyme reactions using HaeIII,HpaI, RsaI or TaqI. Each reaction contained 8 ml PCR product, 1 mlrestriction enzyme, and 1 ml of the appropriate 10� digestionbuffer. All digests were incubated at ambient conditions overnightexcept TaqI, which was incubated overnight at 65 �C. Digest pat-terns for each Pythium isolate were visualized by comparison toa 100 bp ladder following electrophoresis on a 1.5% agarose gelstained with ethidium bromide. Restriction patterns were com-pared to a library of RFLP patterns generated from representativePythium isolates, which had been identified by sequence analysisand morphological characterization in this and previous studies(Mazzola et al., 2002).

2.5. Quantification of soil Pythium populations by real-time PCR

Pythium spp. in soils were quantified by real-time PCR(Schroeder et al., 2006). Briefly, DNA was extracted from soil usinga MoBIO Ultraclean Soil DNA Isolation kit, from two 0.5 g soilsamples per treatment. The DNA was employed in individual 20 mlreactions, conducted in duplicate using FastStart DNA Master SYBRGreen I and a Roche Light Cycler, with conditions and primer pairsdesigned to amplify one of 10 Pythium spp. (Schroeder et al., 2006).After initial analyses, P. paroecandrum, P. aff. echinulatum, P. irreg-ulare Group I, P. ultimum, P. heterothallicum, and P. attrantheridiumprimers were selected for use on all soil treatments.

2.6. Statistical analysis

All statistical analyses were conducted with SAS 9.1 software(SAS Institute Inc., Cary, North Carolina). Data were subjected toanalysis of variance and mean separation was based on FisherProtected LSD. Results were considered significant at P� 0.05.

3. Results

3.1. Weed emergence and biomass in the greenhouse

Seed meal treatments resulted in significant (P< 0.05) re-ductions or increases in plant emergence, with the response beingseed meal or plant dependent. T. aestivum emergence and survivalwere reduced by amendment of soil with B. napus, G. max or S. albaSM, relative to the control (Table 1a). In contrast, pasteurization, B.juncea amendment, and mefenoxam treatments typically increasedplant emergence (Table 1a). Emergence of V. villosa was low overalland consistent treatment effects were not observed, although V.villosa emergence exhibited trends similar to those of T. aestivum inresponse to soil treatments (Tables 1a and 1b). Amendment with S.

Table 1cEffects of seed meal amendments on percent emergence of Echinochloa crusgalliwhen established in two orchard soils

Treatment WVC1a WVC2 TU

5 d 5 d 21 d 5 d 21 d

Control 48 bcb 48 bc 38 cd 27 c 28 efControlþmefenoxam 53 abc 53 bc 54 ab 43 ab 43 abcdPasteurized 56 ab 56 ab 43 bcd 49 a 54 aPasteurizedþmefenoxam 56 ab 56 ab 53 ab 44 ab 47 abcB. napus 51 abc 51 bc 54 ab 41 ab 33 deB. napusþmefenoxam 44 c 44 c 61 a 42 ab 46 abcdB. juncea 65 a 65 a 33 de 33 bc 33 cdeB. junceaþmefenoxam 55 ab 55 ab 53 ab 48 a 40 bcdeS. alba 31 d 31 d 19 f 33 bc 37 bcdeS. albaþmefenoxam 44 c 44 c 46 bc 44 a 51 abG. max 57 ab 57 ab 24 ef 23 c 16 fG. maxþmefenoxam 52 abc 52 bc 47 bc 44 ab 38 bcde

a Orchard designations: WVC, Wenatchee Valley College Orchard, East We-natchee, WA, and Tukey, Tukey Horticulture Research and Experimental Orchard,Pullman, WA. WVC1 represents soil collected in spring 2005, and WVC2 representssoil collected in autumn 2005.

b Means in the same column followed by the same letter are not significantlydifferent (P> 0.05; n¼ 10).

Treatment

bio

mass (g

)

0

10

20

30

40

50

60BroadleafGrass

Control

Fumigated

B. napus

B. juncea

S. alba

ab

b

ab

ab

a

a

ab

b

b

b

Fig. 1. Effect of Brassicaceous seed meal amendments on aboveground weed biomassin an apple planting established in 2005 at the Columbia View Orchard. Values, rep-resented by bars, designated with the same letter are not significantly different(P> 0.05; n¼ 5).

L. Hoagland et al. / Soil Biology & Biochemistry 40 (2008) 1689–16971692

alba SM reduced emergence of E. crusgalli compared to the controlin WVC soils. G. max SM amendment reduced E. crusgalli emergenceonly one time in WVC soils. The majority of pasteurization andmefenoxam treatments, and certain B. juncea amendments, in-creased E. crusgalli emergence relative to the control (Table 1c). Soilamendment with S. alba or G. max SM reduced A. retroflexusemergence in most cases, with most other treatments increasing A.retroflexus emergence (Table 1d). Biomass followed similar trendsto emergence data for all species (not shown).

3.2. Weed emergence and biomass in the field

Soil treatment resulted in statistically significant (P< 0.05) re-duction or increase in weed biomass and T. aestivum emergence,with the response being dependent on seed meal and plastic cov-ering. In the new apple orchard planting established in 2005, B.napus SM amendment resulted in greater yield of grass biomass incomparison to all treatments except fumigation (Fig. 1). In separateplots established at CV in 2005, broadleaf weed biomass was re-duced in all BSM-amended plots covered with plastic relative touncovered plots (Fig. 2). In contrast, without plastic cover, biomass

Table 1dEffects of seed meal amendments on percent emergence of Amaranthus retroflexuswhen established in two orchard soils

Treatment WVC1a WVC2 TU

5 d 5 d 21 d 5 d 21 d

Control 26 abb 30 ab 20 de 26 ab 24 cdefControlþmefenoxam 24 abc 29 abc 29 abcd 24 abc 36 abcdePasteurized 21 abc 33 abc 43 a 21 a 53 abPasteurizedþmefenoxam 26 ab 24 ab 33 abcd 26 abcd 37 abcdB. napus 16 bcd 11 bcd 14 bcd 16 bcd 31 cdefB. napusþmefenoxam 23 abc 43 abc 44 cde 23 a 27 abcB. juncea 23 abc 26 abc 16 ef 23 abc 13 defB. junceaþmefenoxam 37 a 29 abc 31 ab 37 abc 47 abcS. alba 3 d 4 d 6 f 3 d 7 fS. albaþmefenoxam 34 a 30 a 31 ab 34 ab 43 abcdG. max 7 d 10 cd 10 ef 7 cd 13 efG. maxþmefenoxam 23 abc 26 abc 24 cd 23 abc 30 bcdef

a Orchard designations: WVC, Wenatchee Valley College Orchard, East We-natchee, WA, and Tukey, Tukey Horticulture Research and Experimental Orchard,Pullman, WA. WVC1 represents soil collected in spring 2005, and WVC2 representssoil collected in autumn 2005.

b Means in the same column followed by the same letter are not significantlydifferent (P> 0.05; n¼ 10).

from S. alba-amended plots was still lower than the non-treatedcontrol, while biomass from B. napus- and B. juncea-amended plotswas greater than their respective plastic covered plots and control(Fig. 2). Grass biomass followed a similar trend but with no sta-tistically significant differences (Fig. 2). In 2006, though trendswere similar to those observed in 2005, there were no differencesin weed biomass production among treatments (data not shown).However, emergence of planted wheat seeds was reduced in both B.napus and S. alba SM-treated plots relative to the control (Fig. 3);mefenoxam treatment of SM-amended plots eliminated the sup-pression of wheat emergence.

3.3. Pythium soil populations

In greenhouse experiments all soils exhibited significant(P< 0.05) increases in Pythium populations in response to B. napus,S. alba, and G. max SM, with the exception of CV-native soil (Table2). Resident Pythium spp. were not detected in initial samples ofCV-native soil, and SM amendments did not elicit a response in the

Treatment

bio

mass (g

)

0

200

400

600

800

1000

1200

1400

1600BroadleafGrass

Control

Fumigated

B. napus

Plastic

B. napus

Open

B. juncea

PlasticB. juncea

Open S

. alba

Plastic

S. alba

Open

a

ab

bc

a

c

a

bc

bc

a

a

a

a

a aa a

Fig. 2. Effect of Brassicaceous seed meal amendments on above and belowgroundweed biomass in 2005 at Columbia View orchard in field plots not planted to apple.Values, represented by bars, designated with the same letter are not significantlydifferent (P> 0.05; n¼ 5).

Treatment

Em

erg

en

ce

0

2

4

6

8

10

12

Control

Control

+Mefenoxam

B. napus

B. napus

+Mefenoxam

B. juncea

B. juncea

+Mefenoxam

S. alba

S. alba

+Mefenoxam

ab

a

c

bc

bc

ab

c

bc

Fig. 3. Effect of Brassicaceous seed meal amendments on emergence of Triticum aes-tivum seeded in 2006 at Columbia View orchard in field plots not planted to apple.Values, represented by bars, designated with the same letter are not significantlydifferent (P> 0.05; n¼ 5).

Table 3Effect of Brassicaceous seed meal amendments on soil populations of Pythium spp.(cfu g�1 soil) recovered from non-planted experimental plots at Columbia View or-chard, Orondo, WA during 2005

Treatment Pythium

Control 25 ba

Fumigated 63 bB. napus 675 aB. napus-plastic 937 aB. juncea 25 bB. juncea-plastic 0 bS. alba 175 bS. alba-plastic 262 b

a Means in the same column followed by the same letter are not significantlydifferent (P> 0.05; n¼ 5).

L. Hoagland et al. / Soil Biology & Biochemistry 40 (2008) 1689–1697 1693

Pythium spp. population in this soil (Table 2). Pythium spp. pop-ulations reached similar densities after B. napus, S. alba, or G. maxSM amendment, 1216–1916 cfu g�1, in all orchard soils tested, butrelative increases were much lower in TU orchard soil (Table 2). Inall soils, B. juncea amendment resulted in a reduction of Pythiumspp. numbers to near the limit of detection.

In field studies conducted at CV orchard, B. napus SM amend-ment, regardless of tarping, significantly (P< 0.05) elevated soilpopulations of Pythium spp. relative to the control in both 2005(Table 3) and 2006 (not shown). In contrast, Pythium spp. numbersin B. juncea-amended plots were reduced to near zero. Time seriesdata from 2006 revealed an initial Pythium decrease in all BSM-amended plots, followed by rapid increases in B. napus- and S. albaSM-amended soils, with populations reaching their highest in B.napus-amended plots (not shown). For all soil treatments, Pythiumpopulations peaked approximately 8 d post-amendment and thendeclined.

3.4. Pythium root and seed infection

Recovery of Pythium spp. from roots and seeds of all plant typesestablished in WVC2 and TU orchard soils amended with S. albawas significantly (P< 0.05) greater than the control and pasteur-ized treatments as well as the respective SM-amended soils treated

Table 2Effect of seed meal amendments on populations of Pythium spp. (cfu g�1 soil) re-covered from three different orchard soils in greenhouse experiments

WVC1a WVC2 TU CV CV-native

Initial 150 cb 150 c 616 cd 150 d 0 aControl 33 c 67 c 833 c 50 d 0 aB. napus 1300 b 1466 ab 1416 b 1216 a 0 aB. juncea 0 c 0 c 483 d 0 d 0 aS. alba 1350 b 1350 b 1916 a 617 c 0 aG. max 1566 a 1583 a 1516 b 1033 b 0 aPasteurized 0 c 0 c 0 e 0 d 0 a

a Orchard designations: WVC, Wenatchee Valley College Orchard, East We-natchee, WA; Tukey, Tukey Horticulture Research and Experimental Orchard, Pull-man, WA; and CV, Columbia View Orchard, Orondo, WA. WVC1 represents soilcollected in spring 2005, and WVC2 represents soil collected in autumn 2005. CV-native soil was collected in an uncultivated area adjacent to the production orchard.

b Means in the same column followed by the same letter are not significantlydifferent (P> 0.05; n¼ 3).

with mefenoxam (Table 4). Similar results were found in all but onecase with G. max SM-amended soil. In B. napus SM-amended soil,plant infection by Pythium spp. increased in five of eight analyses(Table 4). There was no difference between recovery of Pythiumfrom roots and seeds in B. juncea SM-amended soil and control orpasteurized treatments. Seed and root samples from TU-amendedsoils were infected by P. ultimum, P. attrantheridium and P. hetero-thallicum, whereas plant tissues established in WVC2-amendedsoils were infected primarily by P. irregulare, and P. ultimum. Therewas no preference for a particular Pythium spp. to infect one plantspecies over another.

3.5. Soil Pythium population characterization

Total Pythium populations recovered from WVC2 and TU or-chard soils amended with S. alba, G.max, or B. napus SM were sig-nificantly (P< 0.05) greater than in the control, pasteurized, and B.juncea SM-treated soils (Fig. 4a, b). In both soils, amendment withB. napus SM resulted in Pythium spp. numbers that were lowerrelative to S. alba or G. max SM treatment. Pythium species en-richment varied between the two soil types and between SMs. Forexample, P. irregulare Group I was prominent in WVC2 soil, butabsent in TU soil. In contrast, TU soil amended with S. alba, G. max,or B. napus SM was highly enriched with P. attrantheridium,whereas this species was only slightly enriched by G. max SMamendment in WVC soil. Both soils treated with either B. napus orG. max SM were enriched with P. aff. echinulatum, whereas thisspecies was nearly absent when soil was amended with S. alba SM.

4. Discussion

4.1. Relationship between SM amendment and Pythium on weedsuppression

Application of Brassicaceous plant residues has been promotedas a viable strategy for the control of diverse yield-limiting pests(Lazzeri et al., 2003; Pascual et al., 2004). However, as has been thecase for a variety of bio-based amendments, use of Brassicaceousresidues for control of weeds and soil-borne diseases has not beenwidely adopted due to the inconsistency in performance realizedacross production systems. The ability to determine the underlyingfactor(s) limiting efficacy of these materials in pest control requiresan understanding of the mechanism(s) leading to pest suppression.Although glucosinolate hydrolysis products, and predominantlyisothiocyanates, are generally acknowledged as the primary meansresponsible for the biological activity of Brassicaceous plant resi-dues, recent studies suggest that these chemistries are not the onlyfactors responsible for the observed phytotoxic effects (Boydstonand Hang, 1995; Brown and Morra, 1996; Al Khatib et al., 1997) or

Table 4Effect of seed meal amendment on Pythium spp. infection of root and/or seed (%) using four different seed types in Wenatchee Valley College and Tukey Horticulture Researchand Experimental orchard soils in greenhouse experiments

T. aestivum V. villosa A. retroflexus E. crusgalli

WVC2a TU WVC2 TU WVC2 TU WVC2 TU

Control 0 cb 17 c 22 c 45 b 6 cd 6 b 0 b 0 cControlþmefenoxam 0 c 0 d 6 d 17 c 0 d 0 b 0 b 0 cPasteurized 11 c 0 d 0 d 0 c 0 d 0 b 0 b 0 cPasteurizedþmefenoxam 0 c 0 d 0 d 0 c 0 d 0 b 0 b 0 cB. napus 50 b 100 a 67 b 72 ab 28 bc 46 a 9 b 90 aB. napusþmefenoxam 6 c 28 b 0 d 0 c 0 d 17 b 0 b 6 bcB. juncea 17 c 0 d 28 c 68 ab 11 cd 11 b 0 b 0 cB. junceaþmefenoxam 0 c 0 d 0 d 0 c 0 d 0 b 0 b 0 cS. alba 87 a 100 a 94 a 91 a 80 a 62 a 60 a 0 cS. albaþmefenoxam 6 c 0 d 0 d 6 c 0 d 6 b 0 b 11 bcG. max 64 ab 100 a 94 a 83 a 39 b 45 a 75 a 17 bG. maxþmefenoxam 6 c 0 d 0 d 0 c 0 d 0 b 0 b 0 c

a Orchard designations: WVC, Wenatchee Valley College Orchard, East Wenatchee, WA; Tukey, Tukey Horticulture Research and Experimental Orchard, Pullman, WA; andCV, Columbia View Orchard, Orondo, WA. WVC2 represents soil collected in autumn 2005. CV-native soil was collected in an uncultivated area adjacent to the productionorchard.

b Means in the same column followed by the same letter are not significantly different (P> 0.05; n¼ 3).

Soil treatment

CF

U P

yth

iu

m/g

so

il

0

500

1000

1500

2000

P. attrantheridium

P. heterothallicum

P. ultimum

P. echinulatum

P. irregulare

P. parochandrum

Control

Initial

B. napus

B. juncea

S. alba

G. m

ax

Pasteurized

Soil Treatment

CF

U P

yth

iu

m/g

s

oil

0

500

1000

1500

2000

2500

3000

3500P. attrantheridium

P. heterothallicum

P. ultimum

P. echinulatum

P. irregulare

P. parochandrum

Initial

Control

B. napus

B. juncea

S. alba

G.m

ax

Pasteurized

a

b

Fig. 4. Effect of seed meal amendments on Pythium spp. population resident to We-natchee Valley College (a) and Tukey (b) orchard soil, as determined by real-time PCR.

L. Hoagland et al. / Soil Biology & Biochemistry 40 (2008) 1689–16971694

disease control (Cohen and Mazzola, 2006; Mazzola et al., 2007)attained.

Several lines of evidence from this study demonstrate that weedsuppression in response to certain BSM amendments involvesa microbial mechanism. These include the observation that (i)pasteurization or fumigation of soil prior to sowing of seed im-proved weed emergence in native soils; (ii) the application of theoomycete-selective chemistry mefenoxam reduced the weed con-trol efficacy of most seed meal amendments; and (iii) the non-glucosinolate containing G. max SM provided a degree of weedcontrol that was comparable to S. alba or B. napus SM.

The level of weed control and the effect on Pythium spp. pop-ulations was dependent upon the Brassicaceous species from whichthe seed meal was derived, and in certain instances performance infield trials relative to that obtained in greenhouse trials differedsignificantly. S. alba SM amendment resulted in the greatest andmost consistent weed suppression, although field results were notalways statistically significant. Lack of significance in field trialsmay have been the result of highly variable conditions in terms ofboth weed seed distribution and distribution of Pythium spp. infield soil environments. In contrast, amendment with G. max or B.napus SM also resulted in weed suppression, but results were not asconsistent, and delayed emergence rather than plant death wassometimes observed, with seedling recovery detected during finalassessment of plant emergence 21 d after seeding. Correspond-ingly, soil amendment with S. alba, G. max or B. napus SM signifi-cantly increased Pythium spp. populations. Soil pasteurization ortreatment of SM-amended soils with mefenoxam almost uniformlyincreased plant emergence and biomass. S. alba SM-amended plotstreated with mefenoxam still exhibited some reduction in plantemergence. These results support our hypothesis that plant path-ogenic Pythium spp. mediate, at least in part, the weed suppressionobserved in response to BSM amendments.

Relative to other SM amendments, weed emergence data ob-served in response to B. juncea SM amendment were anomalous. B.juncea SM amendment did not enhance, but rather suppressed,Pythium spp. numbers to near or below the limit of detection,confirming its potential as an alternative treatment for the controlof Pythium spp. (Brown and Morra, 1997; Mazzola et al., 2007).Correspondingly, this amendment did not suppress weed emer-gence in greenhouse trials. In certain instances, enhanced weedemergence was observed in the greenhouse in response to B. junceaSM, again corresponding with the negative impact of the amend-ment on resident Pythium spp. On occasion this same amendmentdepressed weed emergence or biomass relative to the control or

controlþmefenoxam treatment in field trials. It is likely that thisdisparity between greenhouse and field trials with regard to weedsuppression resulted from the experimental design employed. Inthe greenhouse experiments, BSM amendments were applied 4 d

L. Hoagland et al. / Soil Biology & Biochemistry 40 (2008) 1689–1697 1695

prior to sowing weed seeds. As B. juncea SM does not stimulatePythium spp. populations, the likely means of weed control ob-served in this study would be through generation of allylisothio-cyanate (AITC). AITC emission from B. juncea SM-amended soils hasbeen shown to cease within 24 h of seed meal application (Mazzolaet al., 2007). Thus, it is probable that the lack of weed suppressionobserved in our greenhouse trials resulted from the 4-d delay inseeding of soils after B. juncea SM amendment, which circumventedexposure of weed seeds to AITC.

In the instance of S. alba SM amendment, the data indicate thatmultiple mechanisms contributed to the weed suppression ob-served in these studies. Consistent with previous research, we be-lieve that 4-hydroxybenzyl glucosinolate hydrolysis productsproduced after amendment with S. alba SM caused injury to seedsand seedlings (Borek and Morra, 2005). However, S. alba SM appli-cation also resulted in elevated populations of Pythium spp. thatcaused pre- and post-emergence damping-off, which ultimately wasresponsible for seedling death. In contrast, weed suppression fol-lowing G. max or B. napus SM amendment, with zero and low glu-cosinolate content, respectively, likely occurred solely in response toenrichment of and infection by resident pathogenic Pythium spp.

Covering B. napus and B. juncea SM-amended plots with clearplastic resulted in significantly reduced weed biomass relative tothe non-treated control, a response that was not achieved in theabsence of covering treated soils. This finding supports the hy-pothesis that weed suppression resulted in part from release ofvolatile hydrolysis compounds, such as AITC, derived from p-pro-penyl (allyl) glucosinolate, present to a high degree in B. juncea andto small extent in B. napus (Brown and Morra, 1997). However,application of the plastic covering could also have raised soiltemperature creating optimal conditions for growth of Pythiumspp., which exhibit greatest activity in terms of plant infectionduring the spring of the year (Mazzola et al., 2002). This premise issupported by the trend of increased Pythium spp. numbers in B.napus and S. alba SM-amended soils when covered relative to thecorresponding non-covered treatments. Alternatively, it could beargued that covering the soil with plastic resulted in soil solariza-tion, which inhibited weed emergence. However, tarping of soiloccurred in May when temperatures were not high, and in a pre-vious study conducted at this site in 2002, soil temperature undersimilar plastic reached a maximum of 29.4 �C in June and did notexceed 39 �C at a depth of 10 cm during July and August whenannual peak temperatures occur (M. Mazzola, unpublished obser-vations). Had temperatures been high enough (46 �C) to inhibit theseed germination of weeds, such as Amaranthus spp. and E. crusgalli(Stapleton et al., 2000), resident to this site, a corresponding re-duction in Pythium spp. activity also would have been observed.

B. napus SM amendment and 1,3-dichloropropene-chloropicrinfumigation treatments increased weed biomass in some cases. En-hanced availability of nitrogen associated with B. napus SM (Snyderet al., 2006) amendment and the lower enrichment of specific path-ogenic Pythium spp. relative to other SM as observed using real-timePCR analysis likely contributed to this outcome. Greater weed biomassin response to 1,3-dichloropropene-chloropicrin fumigation waslikely due to control of resident Pythium spp. and reduced competitionfrom soil microorganisms for available nutrients. Mefenoxam appli-cation to most BSM-amended and control plots either stimulatedweed emergence or resulted in an increase inweed biomass relative tothe control. Again, these data support our hypothesis that the en-richment of resident Pythium spp. in response to BSM amendmentsplays a significant role in the observed weed suppression.

Findings from these studies demonstrate that optimal efficacy ofBSMs in the control of weeds requires function of the resident soilmicrobial community and specifically the activity of pathogenicspecies of Pythium. Although such a mechanism has the benefit ofutilizing resident soil microbial communities, dependence of weed

suppression on enhancement of resident Pythium spp. may lead toan inconsistency in weed control, such as that reported in previousresearch with Brassicaceous plant residues (Brown and Morra,1997). Quantitatively, Pythium spp. populations varied widelyamong soils and responded differentially to SM amendment.Pythium spp. were not initially detected in CV orchard native soil,and this community did not respond to SM amendment. In con-trast, WVC and TU orchard soils showed a differential response toSM amendments given initial populations, and community en-richment also varied among the different SM amendment types.

Initial Pythium spp. populations were higher in TU soil, whichmay explain why application of B. juncea did not reduce Pythiumspp. populations to near zero, as observed in CV or WVC soil. Basedupon plate count estimates, amendment of all soils with S. alba, B.napus or G. max SM resulted in Pythium spp. enrichment to around1500 cfu g�1, an increase from initial populations of 20–40� in CVand WVC soil, but only 2� in TU soil. Since equivalent amounts ofSM were added to each soil, this could indicate that soils attainedthe maximum Pythium spp. populations capable of being sustainedby the available substrate. Alternatively, the higher clay and OMcontents in TU soil may have exerted a buffering influence thatlimited population expansion and/or reduced the effective avail-able substrate. Similarly, higher clay and OM contents minimize soilacidification that results from nitrification reactions, favoring bac-terial rather than fungal community enrichment in high clay andOM soils (Stotzky, 1986). In addition, recovery of allelopathic phe-nolic compounds varies with soil type (Dalton et al., 1989), andpretreatment of soil to remove organic matter and free metal ox-ides has been found to decrease sorption of phenolic compounds(Cecchi et al., 2004). These different responses to SM amendment indifferent soils may help to explain the variability in weed sup-pression observed under field conditions.

4.2. Pythium community response to seed meal amendment

Total Pythium spp. population estimates were higher using real-time PCR analyses as compared to plate counts. The disagreement inthese data likely resulted from plate counts that onlyaccount for live,active cells. In contrast, real-time PCR is a gene-based approach thatestimates total DNA, which could include that from spores and deadcells. Interestingly, based upon real-time PCR generated data,Pythium spp. populations were lower in soils amended with B. napusSM in comparison to S. alba or G. max SM-amended soils, which maybe a function of the primer sets used. The 10 original primer setswere designed and selected based upon the most prevalent patho-genic Pythium spp. resident in these soils (Schroeder et al., 2006).

Many Pythium spp. resident to agricultural soils are non-path-ogenic to most plant species and can even be beneficial to plantgrowth (Mazzola et al., 2002). It is plausible that B. napus SMamendment resulted in enrichment of a variety of Pythium species,many of which are non-pathogenic or less virulent, and could havecontributed to the lower level of weed inhibition obtained with thisSM relative to G. max or S. alba SM, despite their similar effect ontotal Pythium spp. populations. In a study in which all culturablePythium spp. recovered from SM-treated orchard soil were identi-fied, the population recovered from S. alba SM-amended soil wascomposed primarily of isolates belonging to the species P. irregulareGroup I and P. ultimum var. ultimum, whereas that recovered from B.napus SM-treated soil was dominated by P. heterothallicum (M.Mazzola, unpublished data). P. irregulare Group I and P. ultimum var.ultimum are generally considered to be highly virulent plantpathogens (Chamswarng and Cook, 1985; Mazzola et al., 2002) andcan cause pre- and post-emergence damping-off, whereas P. het-erothallicum is generally a less virulent pathogen of plants and doesnot incite significant damping-off of wheat (Chamswarng andCook, 1985).

L. Hoagland et al. / Soil Biology & Biochemistry 40 (2008) 1689–16971696

4.3. Weed species response to BSM amendment

An additional factor of an agro-ecosystem that may affect efficacyof BSMs is the species composition of the weed seed bank and theirrelative susceptibility to the biological and chemical factors con-tributing to weed suppression. T. aestivum and A. retroflexus weregenerally more susceptible to BSM treatments than were V. villosaand E. crusgalli. Liebman and Davis (2000) speculated that smallweed seeds, like A. retroflexus, may suffer greater allelopathic sus-ceptibility in comparison to large seeds due to their small store ofnutrient and energy reserves, and a greater root length per unitmass, which increases their relative absorptive surface area. How-ever, Haramoto and Gallandt (2005) found monocots to be moresusceptible to allelochemicals than dicots, regardless of seed size.Greater nutrient and energy reserves may enable large dicot seeds totolerate Pythium spp. enrichment and may explain the reduced in-hibition and later recovery observed with the large V. villosa seeds. Inaddition, V. villosa seeds have hard coats, which may help to reduceinfection by Pythium spp. In contrast, the relatively large T. aestivumseed used in our studies exhibited high susceptibility, which mayresult from greater sensitivity as a monocot. Differences in rootingpatterns and seed exudates could also be a factor in the differentialcapacity of Pythium species to suppress individual weed species.

Finally, the capacity of a plant species to escape initial seedmeal-induced suppression has the potential to lead to increasedweed biomass. The function of BSM-induced weed suppressionresulting from Pythium spp. incited pre- and post-emergencedamping-off will not only be dependent upon plant susceptibility,but also the complex of Pythium spp. that resides in any specific soil.Virulence towards a specific plant host varies dramatically (Maz-zola et al., 2002), and individual Pythium species highly virulenttowards one plant species may not cause significant damage toanother plant species (Paulitz et al., 2003). Growth of survivingplants may be enhanced as BSMs are a significant source of nitrogenand phosphorus, and have been used in crop fertilization (Kucke,1993).

4.4. Conclusions

Findings from this study demonstrate that multiple mecha-nisms determine the weed control capacity of Brassicaceous resi-dues and that the mechanisms involved may vary among plantsource. In part, selective enhancement of resident pathogenicPythium spp. contributes to weed control, but this is not true for allBSMs, including B. juncea seed meal. The fact that plant pathogenshave a role in the observed weed control has apparent implicationsfor employing such a strategy in crop production systems, andcaution must be taken in the use of such materials to preventdamage to target crops.

Acknowledgments

We would like to thank the USDA-CSREES Organic and In-tegrated Grant Program and Washington Tree Fruit ResearchCommission for support of this research. Thanks also to SheilaIvanov and Kevin Hansen for technical assistance in lab and fieldexperiments, Kurt Schroeder for assistance with real-time PCR, TimPaulitz and Steve Jones for critical review of the manuscript, andKent Mullinix at WVC orchard and Deb Pehrson at TU for allowingus to collect soil for greenhouse experiments. We acknowledge andthank Mark Evans for assistance in statistical evaluation.

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